Days weaving the lagging strand synthesis of DNA — A personal recollection of the discovery of Okazaki fragments and studies on discontinuous replication mechanism —
- Tsuneko OKAZAKI
- Proceedings of the Japan Academy. Series B…
Nuclease activity persists in the most purified deosyribonucleic acid polymerase preparations from normal and bacteriophage T2-infected Escherichia coli (1,2). It has therefore been difficult to determine whether such a nucleolytic activity is an invariable component of bacterial polymerase. An opportunity to explore a source of polymerase which was promising in this respect was suggested by a survey of a variety of microorganisms for nuclease activity.’ Bacillus subtilis was identified in a group of organisms which have low levels of nuclease activity; since extracts of this organism have substantial polymerase levels, the polymerase to nuclease ratio compared to that in E. coli is relatively high. Purification of DNA polymerase from B. subtilis therefore might yield a nuclease-free enzyme and would also provide a polymerase to compare with the E . coli enzyme for specificity of primers, for relative effectiveness of various base analogues of the deoxynucleoside triphosphates, for synthesis of deosyadenylate-deoxythymidylate copolymer de muo, and for the ability to incorporate a ribonucleotide into a DNA polymer. This report describes the preparation of a polymerase from B. subtilis which has little or no nuclease activity. Studies of the properties of the enzyme reveal fundamental similarities to the E . coli polymerase. Quantitative comparisons of base analogue incorporation support the view that base pairing rather than enzyme specificity determines the incorporation of bases into DNA.